湖泊微拟球藻高含油突变株的构建及筛选  

CONSTRUCTION OF OIL-RICH MUTANTS IN NANNOCHLOROPSIS LIMNETICA

作　　者：张玲香 陈怡雯 胡晗华[1] ZHANG Ling-Xiang;CHEN Yi-Wen;HU Han-Hua(Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;University of Chinese Academy of Sciences, Beijing 100049, China)

机构地区：[1]中国科学院水生生物研究所藻类生物学重点实验室,武汉430072 [2]中国科学院大学,北京100049

出　　处：《水生生物学报》2018年第2期416-420,共5页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金面上项目(41576144)资助~~

摘　　要：研究旨在建立湖泊微拟球藻(Nannochloropsis)的遗传转化体系,然后根据外源基因随机插入的特性,挑取抗性转化子构建突变体库,并从突变体库中筛选高含油突变株。以湖泊微拟球藻自身β-tublin基因启动子和三角褐指藻fcp A终止子驱动和终止来源于细菌的sh ble抗性选择基因,构建了一个转化载体p Pha-T1-TUB。将线性化后的质粒以电穿孔的方法转化湖泊微拟球藻,通过1μg/m L zeocin的抗性平板筛选,并经液体培养基连续传代后,得到了可以稳定遗传的转化子。我们从这些转化子中筛选得到了数株油含量高于野生型,且生长也优于野生型的突变株。其中,2个突变株K26和G5的总脂中多不饱和脂肪酸含量更低,其脂肪酸组成更符合作为生物柴油原料的标准。研究通过随机插入构建突变体库的方法为快速获取优良目的性状的高产油突变株提供了一个有效手段。Nannochloropsis are model species for investigating biofuel production by algae.Many marine Nannochloropsisgenomes have been sequenced recently and their transformation systems have been established.N.limneticais the only freshwater species,and its genome and transformation system are not yet available.In this study,a genetictransformation method for N.limnetica is established and a zeocin-resistance gene is introduced into the genome to obtainmutant library,then high oil-yield mutants are screened from the library.A vector pPha-T1-TUB that has a sh blegene derived by Nannochloropsisβ-tublin promoter and Phaeodactylum tricornutum fcpA terminator was constructed,and the linearized plasmid was introduced to N.limnetica by electroporation.Transformants were able to grow on1μg/mL zeocin.PCR detection indicated that100%of the selected colonies were positive transformants and all resistantcolonies contained sh ble gene after half a year.Several mutants with higher triacylglycerols content and biomasswere obtained.Among them,K26and G5showed lower polyunsaturated fatty acid contents than wild type,whichmeets the requirements of the European Standard EN14214(2004)for biodiesel production.This study provides an efficientmethod to obtain advantageous mutants via generating a library of random resistant gene insertion mutations in N.limnetica.

关 键 词：湖泊微拟球藻 三酰基甘油 电转化 突变株 

分 类 号：Q948.1[生物学—植物学]